OCTOPUS-LIKE 2, a novel player in Arabidopsis root and vascular development, reveals a key role for OCTOPUS family genes in root metaphloem sieve tube differentiation.

Protophloem and metaphloem sieve tubes are essential for transporting carbohydrates and signalling molecules towards sink tissues. OCTOPUS (OPS) was previously identified as an important regulator of protophloem differentiation in Arabidopsis roots. Here, we investigated the role of OCTOPUS-LIKE 2 (OPL2), a gene homologous to OPS. OPL2 expression patterns were analysed, and functional equivalence of OPS and OPL2 was tested. Mutant and double mutant phenotypes were investigated. OPS and OPL2 displayed overlapping expression patterns and a high degree of functional overlap. A mutation in OPL2 revealed redundant functions of OPS and OPL2 in developmental processes in which OPS was known to play a role, notably cotyledon vascular patterning and protophloem development. Moreover, we also uncovered redundant roles for OPS and OPL2 in leaf vascular patterning and, most interestingly, metaphloem sieve tube differentiation. Our results reveal a novel OPS-like protein that, together with OPS, is an important regulator of vascular patterning, root growth and phloem development. OPS and OPL2 are the first genes identified that play a role in metaphloem sieve tube differentiation.

Dnmt1, Dnmt3a and Dnmt3b cooperate in photoreceptor and outer plexiform layer development in the mammalian retina.

Characterizing the role of epigenetic regulation in the mammalian retina is critical for understanding fundamental mechanisms of retinal development and disease. DNA methylation, an epigenetic modifier of genomic DNA, plays an important role in modulating networks of tissue and cell-specific gene expression. However, the impact of DNA methylation on retinal development and homeostasis of retinal neurons remains unclear. Here, we have created a tissue-specific DNA methyltransferase (Dnmt) triple mutant mouse in an effort to characterize the impact of DNA methylation on retinal development and homeostasis. An Rx-Cre transgene was used to drive targeted mutation of all three murine Dnmt genes in the mouse retina encoding major DNA methylation enzymes DNMT1, DNMT3A and DNMT3B. The triple mutant mice represent a hypomorph model since Dnmt1 catalytic activity was still present and excision of Dnmt3a and Dnmt3b had only about 90% efficiency. Mutation of all three Dnmts resulted in global genomic hypomethylation and dramatic reorganization of the photoreceptor and synaptic layers within retina. Transcriptome and proteomic analyses demonstrated enrichment of dysregulated phototransduction and synaptic genes. The 5 mC signal in triple mutant retina was confined to the central heterochromatin but reduced in the peripheral heterochromatin region of photoreceptor nuclei. In addition, we found a reduction of the 5 mC signal in ganglion cell nuclei. Collectively, this data suggests cooperation of all three Dnmts in the formation and homeostasis of photoreceptors and other retinal neurons within the mammalian retina, and highlight the relevance of epigenetic regulation to sensory retinal disorders and vision loss.

Regulation of GABA content by glucose in the chick retina.

Some visual information is processed in the retina by γ-aminobutyric acid (GABA) signaling. Once retinal degeneration and visual impairment caused by diabetic retinopathy (DR) are affecting an increasing number of people worldwide, and the disease is characterized by hyper- and hypoglycemic events, the authors aimed to investigate how retinal GABA cell content is affected by variations in glucose availability. Using the ex vivo chick retinas exposed to different glucose concentrations, we observed that amacrine cells from both inner nuclear layer (INL) and ganglion cell layer (GCL) as well as their processes in the inner plexiform layer (IPL) released GABA through GABA transporter-1 (GAT-1) after 30 min of glucose deprivation. Extending this insult to 60 min triggered a permanent loss of GABA-positive amacrine cells, caused swelling of IPL and cell death. High glucose (35 mM) for 30 min induced an increment in GABA immunolabeling in both outer and inner retina. Further, glucose deprivation effects could not be reverted by basal glucose levels and high glucose did not prevent GABA loss upon a glucose deprivation insult. Therefore, GABA cell content is differently affected by short-term variations in glucose availability. While high glucose modulates outer and inner GABAergic circuits, glucose deprivation affects mainly the inner retina. Also, consecutive alteration in glucose supply was not able to rescue basal GABA content. Therefore, glucose oscillations interfering with GABAergic retinal functioning during early stages of retinopathies should be further investigated.

Developmental expression of dysbindin in Muller cells of rat retina.

Dysbindin, the product of the DTNBP1 gene, was identified by yeast two hybrid assay as a binding partner of dystrobrevin, a cytosolic component of the dystrophin protein complex. Although its functional role has not yet been completely elucidated, the finding that dysbindin assembles into the biogenesis of lysosome related organelles complex 1 (BLOC-1) suggests that it participates in intracellular trafficking and biogenesis of organelles and vesicles. Dysbindin is ubiquitous and in brain is expressed primarily in neurons. Variations at the dysbindin gene have been associated with increased risk for schizophrenia. As anomalies in retinal function have been reported in patients suffering from neuropsychiatric disorders, we investigated the expression of dysbindin in the retina. Our results show that differentially regulated dysbindin isoforms are expressed in rat retina during postnatal maturation. Interestingly, we found that dysbindin is mainly localized in Müller cells. The identification of dysbindin in glial cells may open new perspectives for a better understanding of the functional involvement of this protein in visual alterations associated to neuropsychiatric disorders.

Perivascular, but not parenchymal, cerebral engraftment of donor cells after non-myeloablative bone marrow transplantation.

Myeloablative (MyA) bone marrow transplantation (BMT) results in robust engraftment of BMT-derived cells in the central nervous system (CNS) and is neuroprotective in diverse experimental models of neurodegenerative diseases of the brain and retina. However, MyA irradiation is associated with significant morbidity and mortality and does not represent a viable therapeutic option for the elderly. Non-myeloablative (NMyA) BMT is less toxic, but it is not known if the therapeutic efficacy observed with MyA BMT is preserved. As a first step to address this important gap in knowledge, we evaluated and compared engraftment characteristics of BMT-derived monocytes/microglia using several clinically relevant NMyA pretransplant conditioning regimens in C57BL/6 mice. These included chemotherapy (fludarabine and cyclophosphamide) with or without 2 Gy irradiation, and 5.5 Gy irradiation alone. Each regimen was followed by transplantation of whole bone marrow from green fluorescent protein-expressing wild type (wt) mice. While stable hematopoietic engraftment occurred, to varying degrees, in all NMyA regimens, only 5.5 Gy irradiation resulted in significant engraftment of BMT-derived cells in the brain, where these cells were exclusively localized to perivascular, leptomeningeal, and related anatomic regions. Engraftment in retina under 5.5 Gy NMyA conditions was significantly reduced compared to MyA, but robust engraftment was identified in the optic nerve. Advancing the therapeutic applications of BMT to neurodegenerative diseases will require identification of the barrier mechanisms that MyA, but not NMyA, BMT is able to overcome.

Safety evaluation of INFAT® PLUS: Acute, genetic, teratogenic, and subchronic (90-day) toxicity studies.

INFAT®PLUS, is a sn-2 palmitate enriched fat ingredient intended for infant formula. A battery of toxicological studies was conducted in accordance with the Food Safety Toxicological Assessment GB-15193 (China), to confirm the safety of INFAT®PLUS. In the acute oral toxicity test, the LD50 of INFAT® PLUS was higher than 53.4 g /kg BW and 26.7 g/kg BW for ICR mice and SD rats, respectively. In a subchronic study, INFAT® PLUS was administered by oral gavage to SD rats with maximal daily dose of 8.90 g/kg BW for 90 days. No treatment-related clinical signs or mortalities were observed. The no-observed-adverse-effect level (NOAEL) was set at 8.90 g/kg BW. Similarly, no evidence of genotoxicity effect was noted in several in vitro and in vivo tests, including bacterial reverse mutation (Ames) test, mouse erythrocyte micronucleus test, and chromosome aberration test of mouse spermatogonia/spermatocyte. For the teratogenic evaluations, no toxicological signs were observed in both pregnant SD rat and fetuses, and the NOAEL of INFAT® PLUS was determined to be 8.90 g/kg BW. Based on the obtained results we concluded that INFAT® PLUS was found non-toxic under the experimental conditions, and the totality of the safety data supports its use for infant nutrition.

Retinal Sublayer Analysis in Autoimmune Retinopathy and Identification of New Optical Coherence Tomography Phenotypes.

PURPOSE: Autoimmune retinopathy (AIR) is a poorly characterized disease with a wide phenotypic spectrum, complicating investigations of its underlying pathophysiology. We sought to analyze optical coherence tomography (OCT) retinal thickness changes in AIR patients. METHODS: A retrospective chart review from 2007 to 2017 was performed evaluating AIR patients at a single academic, tertiary referral center. OCT retinal sublayer analysis was performed, and paradoxical thickening phenotypes were reviewed. RESULTS: Twenty-nine AIR patients with positive anti-retinal antibodies and OCT imaging were identified. Overall, AIR patients had thinner retinal sublayers compared to controls; however, 12 patients (41.4%) had paradoxical thickening of the outer plexiform layer (OPL). This revealed two distinct OCT phenotypes. No association was found between retinal sublayer thickness and specific antiretinal antibodies. CONCLUSIONS: While the pathogenicity of antiretinal antibodies remains unclear, the OCT phenotypes observed underscore the potential for identifying clues in the underlying disease processes and clinical diagnosis.

Comparison and enrichment of sn-2 palmitoyl triacylglycerols (OPO/OPL) in fish oil for its potential application as human milk fat substitutes.

Triacylglycerols (TAG) are differences in fatty acid distributions between infant formula and human milk. In this study, fish oil (Tilapia, Golden pompano, Tiger grouper, and Basa) showed the potential as the source of human milk fat substitutes by comparing TAG profiles with infant formula and human milk. The total lipids and TAG of fish were concentrated in the by-products of fish (head and viscera) and contained high levels of palmitic acid, oleic acid, and linoleic acid. Compared with infant formula, fish oil was closer to human milk in sn-2 fatty acid distribution, and sn-2 palmitic acid level in fish oil exceeded 52 % of total palmitic acid, Golden pompano head was the highest (64.46 %). Further research showed that the content of sn-2 palmitoyl TAG (OPO and OPL dominated) increased from 157.16 mg/g TAG to 305.18 mg/g TAG by isopropanol enrichment (solid-liquid ratio: 1:4, temperature: -12 °C, time: 4 h).

Retinal Structure and Function in a Knock-in Mouse Model for the FAM161A-p.Arg523∗ Human Nonsense Pathogenic Variant.

Purpose: Pathogenic variants in FAM161A are the most common cause of retinitis pigmentosa in Israel. Two founder pathogenic variants explain the vast majority of cases of Jewish origin, 1 being a nonsense variant (p.Arg523∗). The aim of this study was to generate a knock-in (KI) mouse model harboring the corresponding p.Arg512∗ pathogenic variant and characterize the course of retinal disease. Design: Experimental study of a mouse animal model. Subjects/Participants/Controls: A total of 106 Fam161a knock-in mice and 29 wild-type mice with C57BL/6J background particiapted in this study. Methods: Homozygous Fam161a p.Arg512∗ KI mice were generated by Cyagen Biosciences. Visual acuity (VA) was evaluated using optomotor tracking response and retinal function was assessed by electroretinography (ERG). Retinal structure was examined in vivo using OCT and fundus autofluorescence imaging. Retinal morphometry was evaluated by histologic and immunohistochemical (IHC) analyses. Main Outcome Measures: Visual and retinal function assessments, clinical imaging examinations, quantitative histology, and IHC studies of KI as compared with wild-type (WT) mice retinas. Results: The KI model was generated by replacing 3 bp, resulting in p.Arg512∗. Homozygous KI mice that had progressive loss of VA and ERG responses until the age of 18 months, with no detectable response at 21 months. OCT showed complete loss of the outer nuclear layer at 21 months. Fundus autofluorescence imaging revealed progressive narrowing of blood vessels and formation of patchy hyper-autofluorescent and hypo-autofluorescent spots. Histologic analysis showed progressive loss of photoreceptor nuclei. Immunohistochemistry staining showed Fam161a expression mainly in photoreceptors cilia and the outer plexiform layer (OPL) in WT mice retinas, whereas faint expression was evident mainly in the cilia and OPL of KI mice. Conclusions: The Fam161a - p.Arg512∗ KI mouse model is characterized by widespread retinal degeneration with relatively slow progression. Surprisingly, disease onset is delayed and progression is slower compared with the previously reported knock-out model. The common human null mutation in the KI mouse model is potentially amenable for correction by translational read-through-inducing drugs and by gene augmentation therapy and RNA editing, and can serve to test these treatments as a first step toward possible application in patients. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Acute macular neuroretinopathy secondary to central retinal artery occlusion.

Purpose: Acute Macular Neuroretinopathy (AMN) may be the result of deep retinal capillary plexus (DCP) impairment, but its mechanism remains elusive. A recent study has described simultaneous onset of Paracentral Acute Middle Maculopathy (PAMM) and AMN, suggesting a related pathogenic pathway. In this report, we analyze and describe the imaging characteristics of patients with concomitant Central Retinal Artery Occlusion (CRAO) and AMN and suggest a mechanistic pathway to explain this relationship. Observations: A total of 2 cases of CRAO, arteritic and non arteritic, were included in this report. At initial presentation, outer retinal layers were intact. At the two-week follow-up visit, both cases displayed Henle fiber layer hyperreflectivity and ellipsoid zone disruption consistent with AMN. Conclusions: Secondary development of AMN in CRAO is a new finding. DCP ischemia secondary to CRAO may lead to Henle fiber layer disruption, leading to the characteristic findings of AMN.

Synthesis of human milk fat substitutes based on enzymatic preparation of low erucic acid acyl-donors from rapeseed oil.

Rapeseed oil has a similar oleic acid/linoleic acid ratio to human milk fat (HMF). However, it can hardly be used for human milk fat substitute (HMFS) synthesis due to high erucic acid content. In this study, Candida cylindracea lipase (CCL) was found to strongly discriminate against erucic acid. Free fatty acids containing low erucic acid and high oleic acid and linoleic acid were prepared from rapeseed oil hydrolysis catalyzed by CCL. The erucic acid content was only 1.58% (initial 8.70%), when the degree of hydrolysis reached 79.58%. The free fatty acids were used as acyl-donors in the acidolysis catalyzed by Novozym 40086. Considering acyl incorporation and migration, the optimum conditions were 1:8 (tripalmitin to acyl-donors), 40 °C and 2 h. The erucic acid content dropped to 0.97% in the HMFS. According to the Q-TOF-MS analysis, the HMFS was rich in 1,3-dioleoyl-2-palmitoyl-glycerol (18.20%) and 1-oleoyl-2-palmitoyl-3-linoleoyl-glycerol (17.96%), which was similar to HMF.

Retinal Thickness and Morphology Changes on OCT in Youth with Type 2 Diabetes: Findings from the TODAY Study.

Objective: To evaluate changes in retinal thickness and morphology using OCT in youth with type 2 diabetes (T2D) and to identify systemic biomarkers correlating with these changes. Design: Retrospective subgroup analysis of a prospective study. Participants: Participants who underwent OCT imaging in the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) trial and its follow-up study TODAY2. Methods: In 2010-2011 (TODAY) and 2017-2018 (TODAY2), 6 × 6-mm macular volume OCT scans were acquired, segmented, and analyzed to generate total retinal thickness, inner retinal thickness, and outer retinal thickness. The main retinal morphologies graded were intraretinal cystoid spaces, subretinal fluid, and posterior vitreous detachment (PVD). Main Outcome Measures: Changes in total and individual retinal layer thickness and development of abnormal vitreomacular morphology between TODAY and TODAY2. Results: Participants had a mean age of 17.9 ± 2.4 years and glycated hemoglobin (HbA1c) of 8.2 ± 2.8% in TODAY and a mean age of 25.0 ± 2.4 years and mean HbA1c of 9.5 ± 2.8% in TODAY2. Longitudinally between assessments, there were overall decreases in outer retinal thickness from 167.2 ± 11.5 microns to 158.4 ± 12.8 microns (P < 0.001) and in photoreceptor thickness from 30.3 ± 2.9 microns to 29.8 ± 4.1 microns (P = 0.04) in the central subfield, while in the inner subfield, we noted a decrease in outer retinal thickness from 150.5 ± 10.1 microns to 144.9 ± 10.5 microns (P < 0.001) and an increase in inner retinal thickness from 136.9 ± 11.5 microns to 137.4 ± 12.6 microns (P = 0.01). Multivariate analysis showed that in the center subfield, HbA1c increases were associated with increases in total retinal thickness (r: 0.67, P = 0.001), whereas fasting glucose was positively correlated with inner retinal thickness (r: 0.02, P = 0.02). In the inner subfield, both systolic (r: -0.22, P < 0.001) and diastolic (r: -0.22, P = 0.003) blood pressures were negatively correlated with total retinal thickness. There was an increase in PVD (18.9%) and cystoid spaces (4.2%). Conclusions: Youth with T2D develop retinal thickness changes on OCT, including increases in total retinal and inner retinal thickness in the center subfield that correlate with HbA1c and fasting glucose, respectively. Taken together with the increased prevalence of abnormal vitreomacular morphology in this cohort at risk, these findings emphasize the importance of controlling risk factors to prevent the development of sight-threatening retinal complications.

Chinese Breast Milk Fat Composition and Its Associated Dietary Factors: A Pilot Study on Lactating Mothers in Beijing.

Regional differences were found in breast milk composition. This study intended to profile the composition of fatty acid (FA) and triacylglycerol (TAG) in Chinese breast milk and to explore its association with maternal diet. Breast milk samples and data of 52 lactating women at 60-90 days postpartum were collected. The FA composition was measured using gas chromatography-flame ionization detection (GC-FID), and the TAG profile was detected by an ultra-performance liquid chromatography system, coupled with accurate-mass quadrupole time-of-flight mass spectrometer. A semi-quantitative food intake frequency questionnaire and a one-time 24-h dietary recall were used to evaluate the previous month's and the short-term dietary intake, including dietary patterns, food groups, and nutrients. Oleic-palmitic-linoleic (OPL) is the most predominant TAG within the Chinese human milk, followed by oleic-palmitic-oleic (OPO), with an average OPL-to-OPO ratio of 1.35. Linoleic acid (LA) and oleic acid (OA) accounted for 23.9 and 32.0% of the total FAs, respectively. Among the food groups consumed during the preceding month, LA content was positively associated with the consumption of soybeans and soybean products (r = 0.311, p = 0.030), whereas a negative correlation was identified with seafood consumption (r = -0.302, p = 0.030). Negative correlations were found between the OA content and the consumption of soybeans and soybean products (r = -0.363, p = 0.009), livestock and poultry meat (r = -0.375, p = 0.006), nuts (r = -0.305, p = 0.028), as well as cooking oil (r = -0.445, p = 0.001). No significant associations were identified between the LA and OA contents and the dietary patterns. This study confirmed a high OPL level in Chinese breast milk and revealed associations of FAs with maternal dietary intake.

Effects of the Major Structured Triacylglycerols in Human Milk on Lipid Metabolism of Hepatocyte Cells in Vitro.

The effect of structured triacylglycerols [1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL), 3-dilinoleoyl-2-palmitoylglycerol (LPL), and 1,3-dioleoyl-2-palmitoylglycerol (OPO)] in human milk on the lipid metabolism was unclear. Hence, this study investigated the effects of different structured triacylglycerols and their mixtures (M) (OPL/LPL/OPO in M1, M2, and M3 were 1.5:0.5:1, 1.2:1.2:1, and 0.5:0.2:1, respectively) on lipid and expression levels of some critical proteins involved in lipid metabolism in LO2 cells. Results showed that there was more lipid accumulation in the LO2 cells exposed to 2,3-dioleoyl-1-palmitoylglycerol (POO) than OPL, LPL, and OPO (p < 0.05), and more lipid accumulation was observed in the OPL group compared to LPL and OPO groups (p < 0.05). Moreover, there was more lipid accumulation in the M3 group compared to M1 and M2 groups. The expression level of diacylglycerol acyltransferase was highest in the POO group compared to LPL, OPO, and OPL groups and was higher in the M3 group than M1 and M2 groups. The expression levels of acetyl-CoA carboxylase 1 and long-chain acyl-CoA synthetase 1 were highest in the OPL group compared to OPO and LPL groups. In comparison to OPO and LPL, OPL seemed to be more likely to increase the content of triacylglycerols and cholesterol in LO2 cells; therefore, whether this was beneficial to the growth and development of infants needs further verification.

Quantitative profiling of glycerides, glycerophosphatides and sphingolipids in Chinese human milk with ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

Human milk lipids are an important energy source and essential nutrients for the growth and development of infants. The UPLC/Q-TOF-MS was used to qualitatively and quantitatively analyze human milk lipids. Totally, 411 species of lipids were identified, in which the content of OPL was generally higher than that of OPO; SM (75.38 mg/L, 40.39%), PE (51.12 mg/L, 27.39%) and PC (40.10 mg/L, 21.49%) had the highest contents among polar lipids, mainly including SM42:2:2 (22.24 mg/L), PE36:2 (C18:0-C18:2, 21.39 mg/L) and PC36:2 (C18:0-C18:2, 19.80 mg/L). In human milk, TAG56:7 (137.14 mg/L), TAG56:8 (59.49 mg/L), TAG58:8 (65.90 mg/L) and TAG58:9 (49.99 mg/L) were the main sources of AA and DHA; PE was an important source of AA and DHA in polar lipids; and linoleic acyl in glycerides and phospholipids had higher contents than other polyunsaturated fatty acyls. These results provided the scientific basis for the simulation of human milk at molecular level.

Comparison of laser microsurgery and open partial laryngectomy for T1-2 laryngeal cancer treatment.

BACKGROUND: This study aims to investigate the clinical efficacy of transoral laser microsurgery and open partial laryngectomy (OPL) in treating T1-2 laryngeal cancer. METHODS: A retrospective analysis was conducted of 182 patients with T1-2 cancer with anterior vocal commissure (AVC) involvement. The local control (LC), disease-free survival (DFS) and overall survival (OS) rates at 5-year follow-up and the influencing factors were analyzed. RESULTS: No significant difference was observed in the LC or DFS rates between the two groups at 3- and 5-year follow-up. No significant difference was found between the two groups with T1-stage disease. The 5-year LC rates were significantly different from patients with grade 3 or 4 tumors on indirect laryngoscopy and patients with class III or IV tumors on the modified Mallampati test (MMT) (log-rank test: χ2=8.037, P=0.005). The 3-year LC rate of OPL in the depth of pathological infiltration (3-5 mm) group was found to be significantly higher than that of TLM. Significant differences in pathological infiltration depth (3-5 mm) existed between the two groups (log-rank test: χ2=5.786, P=0.016). CONCLUSIONS: T1 lesions are generally limited and superficial, and laser surgery can be well-controlled. For patients with difficult airway exposure, surgeons should have extensive surgical experience and skills. It is recommended that a variety of equipment should be ready so that surgeons can convert to open surgery at any time. For patients with a deep infiltration depth, surgeons should examine laryngoscopy imaging results before surgery.

Reduced macular thickness and macular vessel density in early-treated adult patients with PKU.

PURPOSE: Macular structure is poorly evaluated in early-treated phenylketonuria (ETPKU). To evaluate potential changes, we aimed to examine retinas of PKU patients using optical coherence tomography (OCT) with additional OCT angiography (OCTA) and compare the results to healthy controls. METHODS: A total of 100 adults were recruited in this monocentric, case-control study: 50 patients with ETPKU (mean age: 30.66 ± 8.00 years) and 50 healthy controls (mean age: 30.45 ± 7.18 years). Macular thickness, vessel density and flow area of the right eye was assessed with spectral domain OCT angiography SD-OCT(A). Macular microstructural data between the ETPKU and control group was compared. In the ETPKU group, the relationship between visual functional parameters (best corrected visual acuity [VA], spherical equivalent [SE], contrast sensitivity [CS] and near stereoacuity) and microstructural alterations was examined. The dependency of OCT(A) values on serum phenylalanine (Phe) level was analysed. RESULTS: There was significant average parafoveal and perifoveal total retinal layer thinning in ETPKU patients compared to healthy controls (p < 0.016 and p < 0.001, respectively), while the foveal region remained unchanged in the ETPKU group. Whole macular and parafoveal superficial capillary plexus density was significantly decreased in ETPKU compared to controls (p < 0.001). There were no significant differences in the foveal avascular zone, nonflow area, macular superficial and deep capillary plexus between the groups. The temporal parafoveal inner retinal layer thickness was found to negatively correlate with individual Phe levels (r = -0.35, p = 0.042). There was no difference in vascular density and retinal thickness in the subgroup analysis of patients with good therapy adherence compared to patients on a relaxed diet. CONCLUSIONS: Durable elevation in Phe levels are only partially associated with macular retinal structural changes. However, therapy adherence might not influence these ophthalmological complications.

Data on mitochondrial ultrastructure of photoreceptors in pig, rabbit, and mouse retinas.

Photoreceptors are one of the most energy-consuming cell types within the human body. To meet their high energy demand, photoreceptors possess a mitochondrial cluster in the inner segment of the cell. Interestingly, in several species, the inner segment of cone photoreceptors contains extremely large mitochondria that exceed 2 µm in diameter, called mega-mitochondria. We previously reported that pig retinas also contain mega-mitochondria, however, there are few reports whether mega-mitochondria are present in mammalian photoreceptors. In the present experiment, we analyzed pig, rabbit, and mouse photoreceptors under a scanning electron microscope (SEM), and compared the mitochondrial morphology. Our data showed that all three species present numerous mitochondrial clusters in the ellipsoid zone of photoreceptors, adjacent to the outer segment. In the pig retina, the inner segments of cone and rod photoreceptors were localized in different layers; consequently, we were able to distinguish them easily. Mega-mitochondria were identified only in the inner segment of cone photoreceptors. Also, mitochondria of cone photoreceptors, including mega-mitochondria, were dense cristae and high electron-densities compared to those of rod photoreceptors. In the rabbit retina, cone photoreceptors were existed within the layer of rod photoreceptor outer segment. The rod photoreceptors had a characteristic long outer segment. Cone photoreceptors had a short outer segment, and also had a thick inner segment compared to rod photoreceptors. Most of the mitochondria present in the rod photoreceptor inner segment were long and narrow, whereas mitochondria of cone photoreceptors were fragmented and short. Mega-mitochondria was not detected in rabbit retina. In the mouse retina, most of the photoreceptor cells were rod photoreceptors. Since the shape of the inner segments were very similar, we distinguished cone and rod photoreceptors based on the shape of the outer segments. Some mitochondria of both rod and cone photoreceptors were long and narrow, and there was no significant difference in mitochondrial morphology. Our data showed that mitochondrial morphology in the inner segment of photoreceptors vary among mammalian species. Although mega-mitochondria were present in pig photoreceptors, we could not observe their presence in rabbit nor mouse retinas. To our knowledge, this is a first experiment that perform the wide field observation of rabbit and mouse retina using electron microscopy, and that compare the mitochondrial morphology of photoreceptor cells in pig, rabbit and mouse.

Contributions of T cell dysfunction to the resistance against anti-PD-1 therapy in oral carcinogenesis.

BACKGROUND: Programmed death 1 (PD-1) blockade has great effect in the prevention of oral precancerous lesions, but the drug resistance has also been observed. The determinants of immune resistance during the malignant transformation are poorly understood. METHODS: Anti-PD-1 antibody was administered in the 4NQO-induced carcinogenesis mouse models. The mice were then subdivided into PD-1 resistance(PD-1R) group and PD-1 sensitive(PD-1S) group according to the efficacy. The expression of PD-1 and PD-L1, and the abundance of CD3+ T cells in tumor microenvironment between the two groups was tested by immunohistochemistry. In addition, the activation and effector functions, as well as the accumulation of immunosuppressive cells and expression of immune checkpoints of T cells in the draining lymph nodes and spleen between PD-1R and PD-1S group were analyzed by flow cytometry. RESULTS: Our results showed that T cell infiltration in tumor microenvironment, effector T cell cytokine secretion and central memory T cell accumulation in peripheral lymphoid organs were all inhibited in the anti-PD-1 resistance group. Furthermore, we found that an increase of regulatory T cell (Treg) population contributed to the resistance of the anti-PD-1 therapy. Notably, TIM-3 was found to be the only immunosuppressive molecule that mediated the resistance to anti-PD-1 therapy in the oral malignant transformation model. CONCLUSIONS: Our findings identified a novel mechanism that T cell dysfunction contributes to the immune resistance during the malignant transformation of the oral mucosa. This study provides new targets for improving the efficacy of immunotherapy for early stage of tumorigenesis.

Role for Wnt Signaling in Retinal Neuropil Development: Analysis via RNA-Seq and In Vivo Somatic CRISPR Mutagenesis.

Screens for genes that orchestrate neural circuit formation in mammals have been hindered by practical constraints of germline mutagenesis. To overcome these limitations, we combined RNA-seq with somatic CRISPR mutagenesis to study synapse development in the mouse retina. Here synapses occur between cellular layers, forming two multilayered neuropils. The outer neuropil, the outer plexiform layer (OPL), contains synapses made by rod and cone photoreceptor axons on rod and cone bipolar dendrites, respectively. We used RNA-seq to identify selectively expressed genes encoding cell surface and secreted proteins and CRISPR-Cas9 electroporation with cell-specific promoters to assess their roles in OPL development. Among the genes identified in this way are Wnt5a and Wnt5b. They are produced by rod bipolars and activate a non-canonical signaling pathway in rods to regulate early OPL patterning. The approach we use here can be applied to other parts of the brain.